The goal of this proposal for activating the independent phase (ROO) stays the same as that of the original proposal for the award, i.e., to generate a novel and Integrated view of the mechanisms of hematopoietic precursor self-renewal and differentiation using EML (erythroid, myeloid, and lymphocytic) multipotential cells as a model system. EML cells are ideal for studying the molecular control of early hematopoietic differentiation at a large scale. EML cells give rise to the self-renewing CD34+ precursor cells and partially differentiated non-renewing CD34- cells. Large quantities of EML cells can be grown and differentiated in vitro in the absence of an anatomical niche. Based on my K99 phase of study in differential gene expression, and of transcription factor binding using Chip-Sequencing, I hypothesize that there are key regulators in transcriptional regulatory networks determining the choice between EML cell self-renewal and differentiation, such as TCF7 and RUNX1. I have already constructed preliminary transcriptional regulatory circuits regulated by TCF7 and RUNXI. For my ROO phase of research, 1 plan to globally identify the key transcriptional regulators controlling EML cell self-renewal and differentiation by using gene expression and proteomic data to guide the transcriptional regulation work. The binding targets and transcription factors will be assembled into regulatory networks and I will identify target hubs and test for master regulators. Subsequently I will integrate our genomic, proteomics, phosphorylation data and literature into the transcription factor binding networks and further develop a global interaction network. Finally I will confirm key findings in human primary cells. The proposed project can lead to molecular and biochemical studies in my own lab for many years to come.